Diagnosis of sleeping sickness depends on microscopic demonstration of trypanosomes in blood, cerebrospinal fluid (CSF) or lymph node aspirates. Parasitological methods are not easy to use in screening programmes. Development of serological tests for trypanosomiasis has been hindered by the ability of the parasite to keep changing its surface antigenic coat, which allows it to evade the host’s defense mechanisms. FIND is working with partners to develop serological methods based on either antibody or antigen detection that will be specific and sensitive enough to guide treatment.
Figure 1: The new HAT RDT developed by FIND and Standard Diagnostics, plus accessories. Procedure: 20 µl fresh blood from a finger prick is placed into the well of the RDT, followed by 4 drops of diluent. Results can be read after 15 minutes (view demo video). A positive sample has 2 or 3 red bands; a negative has one band (click on picture to view larger image).
Antibody detection test
FIND and Standard Diagnostics (SD) have developed the first lateral flow rapid diagnostic test (RDT) for Trypanosoma brucei gambiense HAT that is cheap and easy to use for screening people (Figure 1). The tests are packed individually and are stable at a temperature of 40°C for up to 25 months; they are performed on fresh blood obtained from a finger prick, and no instrument or electricity is required. The RDT detects host antibodies to infection, and presence of parasites is confirmed by microscopy.
The new RDT is an alternative to the card agglutination test for trypanosomiasis (CATT), the primary screening tool used by control programmes in areas where T.b. gambiense HAT is endemic (Figure 2). Evaluation of prototypes of the new test at sites in Angola, Central African Republic and the Democratic Republic of the Congo (DRC) has been completed, with more than 14,000 participants being tested by both passive and active screening.
The results obtained have confirmed that the new RDT, which is the first ever to be developed for HAT, performs as well as CATT, with the additional advantages of simplicity, and not requiring refrigeration or any instruments. Based on these findings, the design of the product was locked and the test registered (Korean FDA and CE-marking).
Studies to further evaluate the rapid test will be initiated in early 2013 at multiple sites in several endemic countries. The objectives of these studies will be to confirm the accuracy of the test, determine the costs associated with its use, determine the cost-effectiveness of diagnostic strategies that use the rapid test and also determine its ease of use, in comparison to other methods.
The new RDT has been developed with parasite antigens selected through a series of screening activities during the past four years. Scientists and laboratories with antigens that were previously identified as candidate diagnostic probes have been collaborating with FIND in screening native, recombinant and synthetic peptides for their potential in diagnosis of both T.b. gambiense and T.b. rhodesiense, the two forms of sleeping sickness. In order to identify the most promising candidates, an initial panel of 32 different antigens was screened in a dot blot assay by Microcoat in Germany, using a collection of well-defined sera from patients infected with T.b. gambiense and T.b. rhodesiense. The first screen led to the selection of 14 antigens, which underwent two more rounds of screening using dot blot and ELISA, and new collections of positive and negative serum samples, with greater emphasis on specificity. Two of seven antigens delivered to SD were selected for developing the new 1st generation T.b. gambiense RDT.
The RDT will be practical for integration into the horizontal health care system, at health facilities where single format tests such as malaria RDTs and HIV RDTs are already being used. Its inclusion in surveillance programs, in combination with other new methods such as LAMP, could provide an opportunity to develop cost-effective strategies for passive and active screening, thus increasing the coverage of the population at risk and accelerating the process of elimination of the disease.
Meanwhile, FIND and SD have intensified efforts to develop a second generation RDT that, ideally, should be effective for both forms of HAT. In contrast to the first generation test that is made using native antigens generated from pathogenic trypanosomes, efforts are being made to develop a second generation rapid test using recombinant antigens, which would be easier and cheaper to manufacture.
Antigen detection test
Identification of antibodies that are suitable for antigen detection assays and antigens for use in developing antibody detection tests are running concurrently. FIND is working with the Institute of Biotechnology at the University of Brussels, Belgium, to determine the feasibility of using camel heavy-chain antibodies (nanobodies) in tests to detect parasite antigens. A number of promising nanobodies have been identified and are being tested by Standard Diagnostics.
FIND has also worked with the Seattle Biomedical Research Institute (SBRI) to apply the single chain variable fragment (scFv) antibody engineering technology in development of optimized antibody probes for trypanosome antigens in blood. Using a technology called yeast display, high-affinity antibody fragments for a number of T. brucei proteins are generated, and those that are best suited for diagnostic detection in human samples identified. The sensitivity and stability of the probes for the chosen antigens is enhanced further by antibody engineering methods. The outcome will be a set of antibody probes with characteristics of sensitivity, stability, and manufacturability that are superior to probes generated by traditional methods.